The widely accepted steric blocking model of vertebrate skeletal muscle relaxation suggested that myosin subfragment-one (S-1) does not bind to regulated actin (actin + troponin + tropomyosin) in the presence of ATP under relaxing conditions (low Ca++) where ATP hydrolysis is inhibited. We have shown previously by stopped-flow measurements and now by direct binding studies that S-1 binds to regulated actin with about the same affinity in both relaxing and activating conditions. At pH 7.0, 25 degrees and 18 mM ionic strength in activating conditions the rate of ATP hydrolysis is 20 times greater than in relaxing conditions although the association constant of S-1 to regulated actin in the presence of ATP, is about 1.5 x 10 to the fourth M-1 in both cases. We have observed similar behavior at higher (50 mM) ionic strength. Also, we have shown that in relaxing conditions the rate of ATP hydrolysis by S-1 is inhibited because of a large decrease in the V(max). These sata do not support the steric blocking model of muscle relaxation, rather they suggest that in relaxing conditions, the regulatory proteins troponin and tropomyosin inhibit a rate process in the cycle of ATP hydrolysis.